Classical experimental manipulations of developing vertebrate embryos have indicated that there are key signalling centers which regulate the patterning of the animal. Examples of such centers include the ZPA which organizes the anterior-posterior axis of the limb and the notochord and floor plate of the neural tube which organize structures along the main body axis. The gene encoding the key signal produced by these centers has recently been identified (Sonic Hedgehog). The long term objective of this project is to elucidate the role Sonic and related genes play in controlling pattern and to understand the mechanism of their action. The role of Sonic in limb development will be addressed in detail. Sonic will be misexpressed in the anterior of the limb in the absence of an apical ectodermal ridge (AER), and in the presence of an exogenous source of FGF- 4. The endogenous expression of Sonic will be studied in the absence of an AER and in the presence of FGF-4. Expression will also be monitored following infection with a virus carrying a dominant negative FGF- receptor. The exact timing of induction of Hox genes will be determined relative to endogenous Sonic expression and in response to treatment with retinoids. Misexpression of Sonic will be used to explore the function of Sonic in the somite where it may regulate scerotome induction (assayed with a Pax-1 probe) at the expense of myotome (assayed with MyoD), and in left-right asymmetry (assayed by bending of the heart tube). Expression of a membrane-anchored version of Sonic will determine whether diffusion is necessary for the morphogenic signalling. Antibodies against the native sonic protein and epitope-tagged protein will reveal the extent of its normal diffusion. Monitoring the induction of Hox-genes in vitro will be used to determine whether Sonic acts in a concentration-dependent manner. The receptor for Sonic will be isolated by expression cloning and/or yeast 2-hybrid methods and used in studying the mechanism of signalling. Human Sonic will be cloned and used to determine whether defects in this gene are responsible for inherited disorders. A related gene, Indian Hedgehog, is expressed in chondrogenic regions of the developing limb. This gene will be cloned, its detailed expression pattern determined, and its function tested by misexpression.